1. Field of the Invention
The present invention relates to a novel recombinant baculovirus which exhibits quick and potent insecticidal activity, a construction method thereof and an insect pesticidal composition comprising the same.
2. Description of the Prior Art
Baculoviruses are pathogenic to insects, so that they are proliferated to kill the hosts. In addition, baculoviruses are nonpathogenic to the vertebrate and maintain their activity for a long time in fields. Owing to these advantages, as many as 20 viruses are developed as commercially available insecticides. However, baculoviruses have a limited host range and the insecticidal effects are relatively slow. These disadvantages have limited their use as control agents of insect pests.
Recently, active research has been and continues to be directed to the improvement of baculoviruses in usability as specific pest control agents. In this regard, genetic engineering technology provides a useful means therefor. In fact, a number of attempts have been made to produce the baculoviruses which are improved in killing speed, by introducing various foreign genes into the baculoviruses. The foreign genes with this purpose include those coding for Buthus eupeus insect toxin-1 (Carbonell et al., Gene, 73, 409-418 (1988)), Manduca sexta diuretic hormone (Maeda, Biochem. Biophys. Res. Commun., 165, 1177-1183 (1989)), Heliothis virescens juvenile hormone esterase (Hammock et al., Nature, 344, 458-461 (1990); Bonning et al., Insect Biochem. Mol. Biol., 22, 453-458 (1992)), Pyemotes tritici TxP-I toxin (Tomalski and Miller, nature, 352, 82-85 (1991)), Androctonus australis AalT toxin (Stewart et al., Nature, 352, 85-88) and insect-specific spider toxin (Hughes et al., J. Invertebr. Pathol., 69, 112-118 (1997)). Of them, only the gene encoding scorpion toxin allowed the recombinant baculoviruses to show an increased pathogenicity. The killing speed, however, remains slow even in the baculoviruses carrying this gene.
The delta-endotoxin of Bacillus thuringiensis (a gram positive bacteria, hereinafter referred to as xe2x80x9cBtxe2x80x9d), an insect-specific toxin which accumulates in large amounts during the sporulation of Bt, has been demonstrated as an effective means of controlling pest populations by virtue of its potent insecticidal activity and nontoxicity to humans and poultry (Feitelson et al., Bio/Technology, 10, 271-275 (1992)). As for the insecticidal mechanism of the endotoxin, it starts by enzyme cleavage. When taken in by a susceptible insect, the endotoxin is solubilized in the alkaline environment of the gut and cleaved to a smaller active protein by the action of proteases present in the gut juices. The activated protein binds to the epithelial cells of the gut, leading to disruption of the gut. While showing paralytic symptoms owing to the change in the pH of the digestive juice and humor, the susceptible insects cease to eat food and are finally put to death in 24-48 hours. For instance, the lepidoptera specific crystal protein (Cry 1), a kind of Bt endotoxin, is 130-140 kDa in total, but its toxicity is greatly enhanced if the protein is cleaved by proteases to a highly active fragment of about 60-70 kDa which corresponds to the N-terminal half of the protein (Aronson, et al., Microbiol. Rev., 50, 1-24 (1986)).
There were noticeable studies on the improvement of viral insecticidal activity by expressing the Bt endotoxin protein in baculoviruses (Bonning and Hammock, Annu. Rev. Entomol., 41, 191-210 (1996)). However, there was observed no enhancement in pathogenicity of the recombinant baculoviruses although they produce a large amount of the Bt endotoxin proteins in the blood lymphs of the insects. This results from no consideration for the insecticidal mechanism of Bt endotoxin which is fulfilled in the presence of the proteases and the epithelial cells in the gut. That is, the single Bt endotoxin protein which is expressed under the control of the promotor Ppol in the blood lymphs, is not activated as in the gut.
Bearing the activation of the baculovirus polyhedrin by protease in the alkaline environment of the insect gut in mind, the present inventors repeated thorough and intensive research with the aim of developing a novel baculovirus which can kill insect pests with high pathogenicity within a short time and finally, resulted in finding that fusion of Bacillus thuringiensis crystal protein with the baculovirus polyhedrin allows a significant improvement in pathogenicity and killing time.
Therefore, it is an object of the present invention to overcome the above problems encountered in prior arts and to provide a novel recombinant baculovirus which can exhibit pathogenicity within a short time.
It is another object of the present invention to provide a novel recombinant baculovirus which is equipped with a monitoring device for infected insects.
It is a further object of the present invention to provide a construction method of the recombinant baculovirus.
It is still a further object of the present invention to provide a method of constructing a transfer vector for the recombinant baculovirus.
It is still another object of the present invention to provide an insect pesticidal composition which is of potent insecticidal activity and quickness and preventive of overuse.
It is yet another object of the present invention to provide a simple purification method of proteins.
According to an aspect of the present invention, there is provided a recombinant baculovirus, which produces a recombinant polyhedra made up of a baculovirus polyhedrin (PH), a Bacillus thuringiensis crystal protein (CP) and jellyfish Aequorea victoria green fluorescent protein (GFP).
According to another aspect of the present invention, there is provided a construction method of baculovirus transfer vector pColorBtrus, comprising the steps of synthesizing a green fluorescent protein (GFP)-coding DNA fragment from plasmid pGFP by a polymerase chain reaction, synthesizing a polyhedrin (PH) gene from wild-type Autographa californica Nucleopolyhedrovirus by a polymerase chain reaction, inserting the green fluorescent protein-coding DNA fragment and the polyhedrin gene in baculovirus expression vector pAcUW31 in such a way that the 5xe2x80x2-end of the green fluorescent protein-coding DNA fragment is linked to the 3xe2x80x2-end of the polyhedrin gene, to yield baculovirus transfer vector pColorPol, synthesizing a Cry1Ac gene from plasmid pPN6.6 carrying a Bacillus thuringiensis Cry1Ac gene by a polymerase chain reaction, and inserting the Cry1Ac gene between the polyhedrin gene and the green fluorescent protein-coding DNA fragment.
According to a further object of the present invention, there is provided a method of constructing the recombinant baculovirus, comprising the steps of introducing a transfer vector carrying a fusion gene encoding a fusion protein in which a baculovirus polyhedrin, a Bacillus thuringiensis crystal protein and a jellyfish Aequorea victoria green fluorescent protein are directly linked from N-terminal to C-terminal, in sequence, and a wild-type baculovirus into an insect cell, simultaneously, and culturing the cell.
According to a further object of the present invention to provide an insecticidal composition which comprises the recombinant virus.
According to still another object of the present invention, there is provided a purification method of proteins using the transfer vector system.